Microorganisms for the production of tryptophan and process for the preparation thereof

ABSTRACT

A tryptophan producing strain of microorganism is selected from  E. coli  and Corynebacteria and is tryptophan feedback resistant and serine feedback resistant. The serine feedback resistance is by a mutation in a serA allele, where the mutated serA allele codes for a protein which has a K i  value for serine between 0.1 mM and 50 mM. The tryptophan feedback resistance is by a trpE allele which codes for a protein which has a K i  value for tryptophan between 0.1 mM and 20 mM. A process for preparing this microorganism and a process for using this microorganism are disclosed.

The invention relates to microorganisms for the production of tryptophanand to a process for the preparation thereof.

It is known that tryptophan metabolism takes place by a singlebiosynthetic pathway in all micro-organisms hitherto investigated(Somerville, R. L., Herrmann, R. M., 1983, Aminoacids, Biosynthesis andGenetic Regulation, Addison-Wesley Publishing Company, U.S.A.: 301-322and 351-378; Aida et al., 1986, Bio-technology of amino acid production,progress in industrial microbiology Vol. 24, Elsevier SciencePublishers, Amsterdam: 188-206). Tryptophan metabolism, its linkage toserine metabolism, and the genes coding for the principal enzymes aredepicted in FIG. 1.

Known processes for tryptophan production are based on the expression ofa mutated trpE gene which codes for a tryptophan-insensitiveanthranilate synthase together with the other genes of the trp operon ona suitable autonomously replicable vector. Owing to the relatively highcopy number of the genes, there is increased expression of the trp genesand correspondingly an increased amount of the individual enzymes oftryptophan metabolism. This results in overproduction of tryptophan.

Examples of such processes are described for a number of organisms: forexample for E. coli: EP 0 293 207, U.S. Pat. No. 4,371,614, for bacillusU.S. Pat. No. 4,588,687, for corynebacterium and brevibacterium EP 0 338474. A number of problems of process control arise in these processes.There may be instability and loss of the vector or a slowing of growthof the producer strain.

EP-A-0 401 735 (Applicant: Kyowa Hakko Kogyo Co.) describes a processfor the production of L-tryptophan with the aid of corynebacterium orbrevibacterium strains which contain recombinant plasmids. Theseplasmids harbour the genetic information for synthesizing the enzymesDAHP synthase, anthranilate synthase, indole-3-glycerol-P synthase,tryptophan synthase and phosphoglycerate dehydrogenase.Feedback-resistant anthranilate synthase alleles are used.

It is furthermore known to increase tryptophan production in strainswith deregulated tryptophan metabolism by introducing a plurality ofserA, or serA, B, C, wild-type genes. Thus, Chemical Abstracts CA 111(1989) 16 86 88q and CA 111 (1989) 16 86 89r describe the use ofbacillus strains which overexpress respectively the serA wild-typeallele and all wild-type genes of serine metabolism (serA, serB andserC) on plasmids for the production of tryptophan WO-A-87/01130describes the use of serA, serB and serC wild-type alleles for theproduction of tryptophan in E. coli.

Increasing the tryptophan yield by preventing serine degradation in thecell is disclosed in EP-A-0 149 539 (Applicant: Stauffer ChemicalCompany). This patent application describes E. coli K12 mutants in whichthe serine-degrading enzyme serine deaminase (sda) is destroyed. It alsodescribes the use of strains of this type for the production of aminoacids. Example VIII describes the use of a strain of this type for theoverproduction of tryptophan from anthranilate. The explanation for theimproved tryptophan yield compared with a strain with intact serinedeaminase in the European patent application is that, in microorganismsin which the reserve of tryptophan precursors is very high, serine orthe serine biosynthesis capacity is rate-limiting for the production oftryptophan.

The object of the invention was to provide microorganisms which produceincreased amounts of tryptophan and to provide processes which make itpossible to prepare microorganisms of this type.

The object is achieved by strains of micro-organisms which arecharacterised in that they have a deregulated tryptophan metabolism anda serine metabolism which is deregulated at least by onefeedback-resistant serA allele.

For the purpose of the present invention, feedback-resistant serAalleles means mutants of the serA gene which code for a phosphoglyceratedehydrogenase with a serine sensitivity which is less than that of thecorresponding wild-type phosphoglycerate dehydrogenase of the particularmicroorganism.

The combination according to the invention of at least onefeedback-resistant serA allele with a micro-organism with deregulatedtryptophan metabolism results in an increase in the tryptophan yield by,astonishingly, up to 2.6-fold compared with the yield achievable withthe same microorganism without the feedback-resistant serA allele underculturing conditions which are otherwise the same.

The increased production of tryptophan by the strains according to theinvention is unexpected and surprising because feedback-resistant serAalleles show an effect only at a high intracellular serine level (TosaT, Pizer L. J., 1971, journal of Bacteriology Vol. 106: 972-982; WinicovJ., Pizer L. J., 1974, Journal of Biological Chemistry Vol. 249:1348-1355). According to the state of the art (for example EP-A-0 149539), however, microorganisms with deregulated tryptophan metabolismhave a low serine level. This is why no increase in tryptophanproduction is to be expected by the introduction, according to theinvention, of a feedback-resistant serA allele into microorganisms withderegulated tryptophan metabolism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that tryptophan metabolism takes place in all knownmicroorganisms by the depicted metabolic pathway;

FIG. 2 shows diagrammatically the position of the ΔtrpL1 mutation (class2) and of the mutations trpE0, trpE5, trpE6 and trpE8 (class 1);

FIG. 3 shows the sequence of the wild-type SerA gene (SEQ ID NO: 13; SEQID NO: 14);

FIG. 4 shows pGC3 which is the recombinant vector with the SerA gene;

FIG. 5 shows the recombinant plasmid called pGH5;

FIG. 6 shows the plasmid called pKB1321;

FIG. 7 shows the plasmid called pKB 1508;

FIG. 8 shows the recombinant plasmid called pGH11;

FIG. 9 shows the complementing plasmid called pGH5/II;

FIG. 10 shows the complementing plasmid called pGH11/II;

FIG. 11 shows the complementing plasmid called pKB1508/II;

FIG. 12 shows the complementing plasmid called pGH5/III; and

FIG. 13 shows the resulting vector pGC3/I which is used to transformCorynebacterium glutamicum ATCC21851.

Since tryptophan metabolism takes place in all known microorganisms bythe metabolic pathway depicted in FIG. 1, and the techniques to be usedfor preparing the strains according to the invention are known inprinciple and can be applied to all microorganisms, strains according tothe invention can be prepared from any desired microorganisms.

Suitable and preferred for the preparation of a strain according to theinvention are bacteria. Gram-negative bacteria, especially E. coli, aresuitable and particularly preferred.

Strains according to the invention can be obtained by entirely orpartially abolishing the regulation of tryptophan metabolism in adesired tryptophan-prototrophic initial strain, and introducing afeedback-resistant serA allele into this strain.

Strains according to the invention can likewise be obtained by restoringthe ability to synthesise tryptophan in tryptophan-auxotrophic initialstrains, with deregulation of the restored tryptophan metabolism, andintroducing a feedback-resistant serA allele into strains of this type.

Deregulation of tryptophan metabolism in micro-organisms is possible bya number of different processes which are known from the state of theart.

One possibility for deregulation of tryptophan metabolism is to modifythe enzyme anthranilate synthase. This enzyme catalyses the first stepin the tryptophan-specific biosynthetic pathway in all microorganisms.Its activity is inhibited by tryptophan, and it thus regulates,depending on the amount of tryptophan, the flow of metabolites throughthe tryptophan biosynthetic pathway. The enzyme is encoded by the trpEgene.

Mutated trpE genes which code for anthranilate synthases with atryptophan sensitivity which is less than that of the correspondingwild-type anthranilate synthase, which are also calledfeedback-resistant trpE alleles hereinafter, can be obtained bymutagenesis and subsequent selection of a tryptophan-prototrophicinitial strain. To do this, the relevant strain is subjected to atreatment which induces mutations (Miller J. H., 1972, Experiments inMolecular Genetics, Cold Spring Habor [sic] Laboratory, U.S.A.:113-185).

The treated strain is cultured on a nutrient medium which contains atleast one tryptophan antagonist in an amount sufficient to inhibitgrowth of the strain. Examples of suitable tryptophan antagonists are4-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan, halogenatedtryptophans, tryptazan, indole and indoleacrylic acid.

Resistant clones are tested for the tryptophan sensitivity of theiranthranilate synthase. The tryptophan sensitivity of the anthranilatesynthase can be determined by any method which permits the activity ofthis enzyme to be determined in the presence of tryptophan. For example,chorismate can be reacted in a suitable buffer system with glutamine,which is its partner in the reaction, under enzyme catalysis (Bauerle R.et al., 1987, Methods in Enzymology Vol. 142: 366-386). Aliquots wereremoved from the assay mixture kinetically, and the amount of thereaction product anthranilate produced per unit time was determined byHPLC analysis. The amount of anthranilate produced per unit time is adirect measure of the activity of anthranilate synthase. The assay iscarried out in the presence and absence of tryptophan in order todetermine the sensitivity of the anthranilate synthase assayed.

It is equally possible to generate tryptophan-insensitive trpE allelesby direct genetic manipulation (Bauerle R. et al., 1987, Methods inEnzymology Vol. 142: 366-386). A number of mutations in the amino-acidsequence of anthranilate synthase leading to a reduced sensitivity ofthe enzyme to tryptophan have been described for various organisms. (Forexample for Salmonella: Caliguiri M G., Bauerle R., 1991, J. of Biol.Chem. Vol. 266: 8328-8335; fur brevibacterium, corynebacterium: MatsuiK. et al., 1987, J. Bact. Vol. 169: 5330-5332).

There are known methods which make it possible to introduce a mutationat a specific point in a DNA fragment. Methods of this type aredescribed, inter alia, in the following publications:

Sakar G., Sommerauer S. S., 1990, Bio Techniques 8: 404-407, describepolymerase chain reaction-dependent site-directed mutagenesis;

Ausubel F. M. et al., 1987, Current Protocols in Molecular Biology,Greene Publishing Associates, describes [sic] phage M13-dependentmethods;

Smith M., 1985, Ann. Rev. Genet. 19: 423-462, describes other methods.

The DNA fragment which embraces the wild-type trpE gene is recombined ona vector using previously described standard techniques for preparingrecombinant DNA. Application of the abovementioned methods to thesite-directed mutagenesis results in modification of one or morenucleotides in the DNA sequence so that the amino-acid sequence nowencoded by the gene corresponds to the amino-acid sequence of atryptophan-insensitive anthranilate synthase. The described techniquescan be used to introduce into any desired trpE gene one or moremutations which result in the encoded anthranilate synthase having anamino-acid sequence which leads to tryptophan insensitivity.

In addition, the following properties are desirable but not absolutelynecessary in the strain according to the invention: a defectivetryptophan repressor protein, a defective attenuation control ofexpression of the trp operon, and a defective tryptophanase. Theseproperties can be obtained in the strain according to the invention mostsimply by choosing an initial strain which already has one or more ofthe appropriate properties. The preparation or choice can be carried outby a combination of the selection methods mentioned hereinafter.

The tryptophan repressor protein is a major regulatory protein oftryptophan biosynthesis. Together with tryptophan as aporepressor, thisprotein represses the expression of the trp operon genes. The protein isencoded by the trpR gene. Tryptophan repressor mutants can be selected,for example, from among mutants which are resistant to tryptophanantagonists such as, for example, 5-methyltryptophan. Examples aredescribed in J. Mol. Biol. 44, 1969, 185-193 or Genetics 52, 1965,1303-1316.

Besides control by the trpR-encoded protein, the trp operon isadditionally subject to attenuation control. A DNA region in front ofthe first gene of the trp operon is responsible for regulation.Mutations or deletions in this region may lead to deregulation. Mutantsof this type can be selected from among mutants which are resistant totryptophan antagonists such as 5-methyltryptophan. On the other hand,mutations of this type, but especially deletions, can be obtained bysite-specific induction of this modification, by methods ofsite-directed mutagenesis, in the attenuation region of the DNA. It ispossible, by the techniques already described of site-specificmutagenesis, to recombine the inactivated attenuator region into thechromosome of the strain according to the invention in place of thenatural attenuator region.

The enzyme tryptophanase (tnaA) catalyses the degradation of tryptophanto indole, pyruvate and NH₃. It is desirable for this enzyme to beinactive in tryptophan producer strains. Strains deficient in thisenzyme can be obtained by subjecting the organisms to a mutagenictreatment, and seeking from among the mutants those which are no longerable to utilise tryptophan as a source of carbon and nitrogen. Adetailed example is described in J. Bact. 85, 1965, 680-685.Alternatively, it is equally possible to introduce into the tnaA gene,using the abovementioned techniques, site-specific deletions whichresult in inactivation.

A number of additional mutations of the initial strain are suitable forbringing about a further increase in tryptophan production. Thus, it ispreferable for not only the tryptophan biosynthetic pathway but also thegeneral aromatic amino acid biosynthetic pathway (shikimic acid pathway)to be regulation-insensitive. This is why strains which have aregulation-insensitive dehydroarabinoheptulusonate [sic] synthase andwhose tyrosine repressor protein (tyrR) is inactivated by a mutation ordeletion are preferred as initial strains for preparing strainsaccording to the invention. Equally preferred are strains whosephenylalanine and tyrosine metabolism is impaired. This ensures that theflux of the precursor molecule chorismate is exclusively in tryptophan.Strains of this type have, for example, mutations or deletions in thepheA and/or tyrA genes.

A number of strains are known to be deregulated in one or more steps oftryptophan biosynthesis or the shikimic acid pathway and to overproducetryptophan. Examples are Bacillus subtilis FermBP-4, FermP1483 (DE3123001), Brevibacterium flavum ATCC 21427 (U.S. Pat. No. 3,849,251),Corynebacterium glutamicum, ATCC 21842-21851 (U.S. Pat. No. 3,594,279,U.S. Pat. No. 3,849,251), Micrococcus luteus ATCC 21102 (U.S. Pat. No.3,385,762), E. coli ATCC 31743 (CA 1182409). These strains are likewisesuitable as initial strains for preparing the strains according to theinvention. They show that tryptophan producer strains according to theinvention can be obtained in a wide variety of organism groups.

Besides the strains with deregulated tryptophan metabolism, required forthe preparation of the strains according to the invention is at leastone gene which codes for a phosphoglycerate dehydrogenase whose serinesensitivity is less than that of the corresponding wild-typephosphoglycerate dehydrogenase.

Phosphoglycerate dehydrogenase (PGD) is encoded by the serA gene. Thesequence of the wild-type serA gene is known (Tobey K. L., Grant G. A.,1986, J. Bac. Vol. 261, No. 26: 1279-1283). Overexpression of thewild-type serA gene product via a plasmid vector is likewise known(Schuller et al., 1989, J. Biol. Chem. Vol. 264: 2645-2648).

The preparation of feedback-resistant serA alleles using classicalgenetic methods is described by Tosa T., Pizer L. T., 1971, J. Bac. Vol.106, No. 3: 972-982. In that case selection took place via theresistance of the mutants to the serine analogue serine hydroxamate. Themutations were not characterised in detail in this publication; theeffect of the mutation on metabolism was not investigated.

Feedback-resistant serA alleles can also be obtained, for example, bysubjecting a microorganism to mutagenesis. Suitable mutagens are UVlight and any chemical mutagens such as, for example, ethylmethane-sulphonate or N-methyl-N′nitro-N-nitrosoguanidine. The dosageand exposure time are determined by conventional methods for the chosenmutagen (Miller J. H., 1972, Experiments in Molecular Genetics, ColdSpring Harbor Laboratory, U.S.A.: 113-143).

Mutagen-treated organism populations are subjected to selection forclones with serA genes which code for serine-insensitivephosphoglycerate dehydrogenases. For example, a mutagen-treatedpopulation is incubated on solid growth medium which contains serinehydroxamate in an amount sufficient to inhibit the growth ofnon-resistant bacteria. Resistant clones are assayed for the serinesensitivity of their phosphoglycerate dehydrogenase. One embodiment ofthis method is described by way of example by Tosa and Pfizer, 1971, J.Bact. 100, 3: 972-982.

Alleles which code for a serine-insensitive phosphoglyceratedehydrogenase can likewise be generated by genetic engineeringtechniques.

The region of the PGD which mediates the serine regulation is located inthe C-terminal region of the protein. This is why insertions,substitutions or deletions of one or more amino acids are preferablyintroduced in the C-terminal 25% of the PGD protein, particularlypreferably in the 50 C-terminal amino acids of the PGD protein. Theseresult in a reduced sensitivity of the PGD to serine.

Alleles which code for PGDs of this type are obtained by modifying the3′ region, which codes for the said C-terminal regions of the PGD, ofthe serA gene. To do this, the unmutated serA gene is recombined on acloning vector by using the techniques known to the person skilled inthe art for preparing recombinant DNA, such as restriction, ligation andtransformation (Maniatis T., Fritsch E. F. and Sambrook J., MolecularCloning: A Laboratory Manual 1982, Cold Spring Harbor Laboratory).Specific modifications in the 3′ region of the structural gene can beachieved, for example, by techniques of site-directed mutagenesis.

Examples of serine-insensitive PGDs which are suitable for expression inmicroorganisms with deregulated tryptophan metabolism are listed inTable 1 by depicting their C-terminal amino-acid sequence. Apart fromthe depicted region, the protein sequences of the enzymes do not differfrom the wild-type sequence.

The following assays were used to test the gene products of the serAalleles for PGD activity and serine sensitivity:

The PGD activity was determined by detection of the forward or reversereaction of the enzyme by the method of McKitrick, J. C. and Lewis J.P., 1980, J. Bact. 141: 235-245. The enzyme activity is measured in thiscase without serine and with various concentrations of serene. The saidassay is suitable for determining the serine sensitivity of anyphosphoglycerate dehydrogenase. It is likewise possible to employ anyother method for measuring the PGD activity.

The measure used for the serine sensitivity of the enzyme is the K_(i)value, that is to say the serine concentrations which inhibit theactivity of the enzyme by 50%. The K_(i) values and C-terminalamino-acid sequences of a number of feedback-resistant serA alleles andof the wild-type serA gene (serAWT) are listed in Table 1.

TABLE 1 C-terminal amino-acid sequences of the mutated SerA allelesK₁/mM ser AWT SEQ ID NO: 1 0.02 serA 5 SEQ ID NO: 2 0.2 serA 1508 SEQ IDNO: 3 3.8 serA 11 SEQ ID NO: 4 50 serA 1455 SEQ ID NO: 5 100

Suitable and preferred for preparing the strains according to theinvention are, surprisingly, serA alleles with a K_(i) value between 100μM and 50 mM serine.

It is possible to employ for expression of the PGD proteins in thestrain according to the invention any recombinant vector which leads toexpression of the serine-insensitive serA alleles. A recombinant vectorsuitable for preparing the strains according to the invention comprisesat least one serA gene which codes for a PGD which has a serinesensitivity which is less than that of the wild type, and a vectorportion which is autonomously replicable in the recipient strain.

Examples of vectors which are autonomously replicable in E. coli arelisted in Pouwels P. H., Enger-Valk B. E., Brammar W. J. 1985, CloningVectors, Elsevier, Amsterdam. Vectors of this type include:

Plasmids with a high copy number such as, for example, pBR322; pUC12,

Plasmids with an intermediate copy number such as, for example,pACYC184,177,

Plasmids with a low copy number such as, for example, pSC101,

Phage vectors such as, for example, M13, λ vectors.

Comparable vectors are described for a large number of bacteria (forexample EP 0 401 735 for corynebacteria and brevibacteria or in CA 111(1989) 168688q).

Suitable and preferred are vectors with an intermediate to low copynumber; vectors with a p15A replicon, such as pACYC184 (ATCC37033) orpACYC177 (ATCC37031), are particularly preferred.

A large number of vectors for other bacteria are described in theliterature (Pouwels et al., 1985, Cloning Vectors, Elsevier SciencePublishers, Amsterdam).

Suitable recombinant vectors can be generated by standard techniques forpreparing recombinant DNA. These techniques are described in detail, forexample, in Maniatis T., Fritsch E. F. and Sambrook J., 1982, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, U.S.A. orAusubel F. M. et al., 1987, Current Protocols in Molecular Biology,Greene Publishing Associates, U.S.A.

Preparation of recombinant vectors is possible, for example, byfragmenting, using restriction enzymes, the DNA of a donor organismwhich has in the chromosome or on a recombinant vector a serA allelewhich codes for a serine-insensitive PGD. The fragmented DNA is ligatedby conventional methods, for example by using the enzyme T4 DNA ligase,into a vector molecule which has likewise been linearised by restrictionenzymes. The ligation mixture is used to transform recipient strainswith deregulated tryptophan metabolism by known processes, such ascalcium chloride shock or electroporation. Vectors which contain therequired serA alleles can be obtained in recipient strains, for example,by the abovementioned methods such as selection for antibioticresistance or complementation of serA mutations.

In another embodiment of the strains according to the invention, theserA alleles are integrated as single copy into the chromosome. This canbe achieved, on the one hand, by carrying out the mutagenesis andselection strategies described above directly with atryptophan-deregulated initial strain. It is also possible, on the otherhand, to integrate serA alleles which are present on recombinant vectorsinto the chromosome of the producer strain. A number of methods forintegration of this type are known. Descriptions of these techniques areto be found in the following publications:

Phage Lambda-mediated Integration: Balakrishnan and Backmann, 1988, Gene67: 97-103; Simons R. W. et al., 1987, Gene 53: 85-89;

recD-dependent gene replacement: Shervell et al., 1987, J. Bact. 141:235-245;

Other methods: Silhavy et al., 1988, Experiments with Gene Fusions, ColdSpring Harbor Laboratory.

Fermentation of strains according to the invention revealed, completelysurprisingly, that strains containing a feedback-resistant serA allelewith a K_(i) value for serine between 100 μM and 50 mM and a deregulatedtryptophan metabolism, containing a trpE allele with a K_(i) value fortryptophan between 0.1 mM and 20 mM, provide the highest yield oftryptophan.

The following examples serve to illustrate the invention further.

EXAMPLE 1 Screening for Feedback-Resistant trpE Alleles and Integrationof These Alleles Into the Chromosome

The tryptophan analogue 5-methyltryptophan was employed for the searchfor feedback-resistant trpE alleles.N-Methyl-N′-nitro-N-nitroso-guanidine (NG) was used as mutagenic agent.The initial strain used was E. coli K12 YMC9 ATCC33927. The mutagenesisprocedure was based on the data of Miller (Miller J. H., 1972,Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.: 125-129).

About 2×10⁹ cells of YMC9 in the exponential phase of growth from aculture grown in LB were incubated with 50 μg/ml NG in 4 ml of 0.1 M Nacitrate buffer pH 5.5 at 37° C. for 30 min. After two washes with 0.1 Mphosphate buffer pH 7.0, 0.1 ml of cells was incubated in LB shaking at37° C. overnight. Subsequently, 0.1 ml of cells from various dilutions(10⁻³, 10⁻⁴, 10⁻⁵ in 0.9% NaCl) were placed on minimal medium platescontaining 100 μg/ml 5-methyltryptophan. Besides 5-methyltryptophan, theminimal medium contained 5 g/l glucose, 5 mg/l vitamin B1, 3 g/l KH₂PO₄,12 g/l K₂HPO₄, 0.3 g/l MGSO₄×7H₂O, 0.1 g/l NaCl, 5 g/l (NH₄)₂SO₄, 14.7mg/l CaCl₂×2H₂O, 2 mg/l FeSO₄×7H₂O, 1 g/1 Na₃ citrate and 15 g/l agar.After 24-48 h at 37° C., 5-methyltryptophan-resistant clones were pickedout and plated out on the above plates.

The resulting mutants were characterised by determining the K_(i) valueof the trpE gene product for tryptophan (Bauerle R. et al., 1987,Methods in Enzymology Vol. 142: 366-386). It was possible in this way todivide the mutants into two classes. Class 1 mutants hadfeedback-resistant anthranilate synthases. Class 2 mutants had enzymeswith increased anthranilate synthase activity and with an unchangedK_(i) value.

For characterisation at the molecular level, the relevant DNA regions ofthe various mutants were cloned and sequenced. For this purpose, thechromosomal DNA was isolated in each case and cleaved with therestriction enzymes NheI and ClaI in one mixture. Fragments with a sizeof about 5 kb were isolated and ligated to the NheI/Clal pBR322 fragmentwhich is 4158 bp in size. The ligation mixture was transformed into atrpE strain of E. coli KB 862 (DSM 7196). Clones which were able to growon minimal medium without tryptophan were selected. The complementingplasmids all contained a 5 kb NheI/ClaI fragment. Besides the trpE andtrpD genes, this 5 kb NheI/ClaI fragment also contains DNA regionsupstream from trpE (about 0.8 kb) and downstream from trpD (about 1 kb).Table 2 lists the amino-acid sequence differences and the K_(i) valuesof 4 Class 1 mutants. The sequences of the mutants agree in the regionswhich are not depicted with the wild-type sequence.

TABLE 2 Amino-acid sequences of the mutated trpE alleles Kj/mM trpE WTSEQ ID NO: 6 0.01 trpE 0 SEQ ID NO: 7 0.1 trpE 5 SEQ ID NO: 8 3.0 trpE 6SEQ ID NO: 9 <15 trpE 8 SEQ ID NO: 10 15

Sequence analysis of the class 2 mutants showed that mutations werepresent either in the operator region of the trp promoter or in the DNAregion which codes for the trp leader peptide. The mutations calledΔtrpL1 and ΔtrpL2 have a deletion which is 136 bp and 110 bp,respectively in size in the DNA region which codes for the leaderpeptide. The deletion embraces in the ΔtrpL1 mutation the region fromnucleotide position 33 to position 168, and in the ΔtrpL2 mutation theregion from nucleotide position 11 to 120 in the sequence stored in theEMBL data bank under AC number V00372.

In order to achieve stronger expression of the feedback-resistant trpEalleles, the two mutant classes were combined. The class 2 mutationΔtrpL1 was used for this. FIG. 2 shows diagrammatically the position ofthe ΔtrpL1 mutation (class 2) and of the mutations trpE0, trpE5, trpE6and trpE8 (class 1).

The 1.6 kb NruI fragment which harbours the ΔtrpL1 mutation was isolatedfrom the plasmid pΔtrpL (FIG. 2) and exchanged for the correspondingwild-type NruI fragment of the plasmids pE0, pE5, pE6 and pE8 (FIG. 2).The resulting plasmids were called pIE0, pIE5, pIE6 and pIE8respectively, and were used for chromosomal integration by homologousrecombination. To do this, the chromosomal NheI/ClaI fragment which isabout 5 kb in size from each of the said plasmids was isolated from lowmelting agarose as described in Example 2 and transformed in linear forminto the recD strain PD106 [ΔtrpLD102]. The transformation method usedwas the CaCl₂ method of Cohen et al., 1972, Proc. Natl. Acad. Sci.U.S.A. 69: 2110-2114. The strain PD106 was deposited in accordance withthe Budapest treaty on 28.07.1992 at the Deutsche Sammlung f{umlaut over(u)}r Mikroorganismen (DSM) under number 7195 (DSM 7195) the DSM islocated at Mascheroder Weg 1B, D-3300 Braunschweig, Germany. Cloneswhich were able to grow on minimal medium without tryptophan and wereampicillin-sensitive, that is to say plasmid-free, were selected. ThetrpE alleles which code for variously feedback-resistant trpE enzymesand are each combined with the ΔtrpL1 mutation were transferred from therelevant strains into KB862 by P1 transduction (Miller J. H., 1972,Experiments in Molecular Genetics. Cold Spring Harbor, N.Y.: 201-205).The strain KB862 was deposited in accordance with the Budapest treaty on28.07.1992 at the Deutsche Sammlung f{umlaut over (u)}r Mikroorganismen(DSM) under number 7196 (DSM 7196). Selection was for growth ontryptophan-free minimal medium. The resulting strains were called PD103(trpE0), KB862 (trpE5), SV164 (trpE8) and SV163(trpE6).

EXAMPLE 2 Preparation of serA Genes Which Code for Serine-InsensitivePhosphoglycerate Dhydrogenases [sic].

The serA wild-type gene was cloned from the E. coli strain E. coli B(ATCC 23226) on the plasmid vector pUC18.

In order to obtain the chromosomal DNA of this strain, it was culturedin Luria broth at 37° C. overnight. The bacterial cells were harvestedby centrifugation (4000 g). Lysis of the cells and purification of theDNA were carried out by the protocol described by Ausubel et al., 1987,2.4.1-2.4.2, Current Protocols in Molecular Biology, Greene PublishingAssociates. The amount of DNA obtained was determined byspectrophotometry at a wavelength of 260 nm. The yields were around 600μg/100 ml.

10 μg of the chromosomal DNA were cleaved with the restriction enzymeSphI (Boehringer Mannheim GmbH) under the conditions stated by themanufacturer. About 3 μg of the fragment mixture were ligated to 0.2 μgof the autonomously replicable plasmid vector pUC18 (supplied byBoehringer Mannheim GmbH), which had likewise been cut with SphI, by theenzyme T₄ ligase (supplied by Boehringer Mannheim GmbH) under theconditions prescribed by the manufacturer. The ligation mixture was usedto transform the serA mutant PC1523 (CGSC#:5411;) (CGSC: E. coli GeneticStock Center, Department of Biology 255 OML, Yale University, Postbox6666, New Haven, Conn., U.S.A.). The transformation method used was theCaCl method of Cohen et al., 1972, Proc. Natl. Acad. Sci . U.S.A. 69:2110-2114. The transformed bacteria were plated out on minimal mediumwithout serine. Clones which grew without serine contained the serA genefrom E. coli or an SphI fragment which is 3.5 kb in size. The sequenceof the wild-type SerA gene is depicted in FIG. 3 (SEQ ID NO: 13; SEQ IDNO: 14). The recombinant vector with the SerA gene was called pGC3 (FIG.4).

The serA allele serA5 was prepared by cutting the plasmid pGC3 with therestriction enzymes SalI and KpnI (supplied by Boehringer Mannheim GmbE)in accordance with the manufacture's data. The resulting fragments werefractionated by agarose gelectrophoresis [sic]. The SalI-KpnI fragmentwhich is 2.0 kb in size and which contains the complete serA gene apartfrom the 8 C-terminal codons was purified from the gel. To do this, theelectrophoresis was carried out on low-melting agarose (supplied byBoehringer Mannheim GmbH) so that it was possible to recover the DNAsimply by melting the agarose. 0.2 μg of this fragment were ligated withequimolar amounts of a HindIII/SalI-cut pUC18 and of a syntheticallyprepared, double-stranded oligionucleotide by T₄ ligase (supplied byBoehringer Mannheim GmbH) in accordance with the manufacture's data. Thenucleotide sequence of this oligonucleotide is as follows.

SEQ ID NO: 11

This oligonucleotide makes up 7 of the 8 last C-terminal codons of theserA gene. The stop triplet TAA is introduced in place of the eighthcodon. The phosphoglycerate dehydrogenase encoded by this SerA gene isthus truncated by one C-terminal amino acid. The amino-acid sequence ofthe mutated PGD is shown in Table 1 (serA5). The recombinant plasmid wascalled pGH5 (FIG. 5). The serA mutant PC1523 was transformed with theligation mixture.

The serA allele serA1508 was prepared as follows. The plasmid pGC3 wascut with SphI/SalI (supplied by Boehringer Mannheim GmbH) in accordancewith the manufacture's data A 3 kb fragment which harbours the completeserA gene was purified by gel electrophoresis and ligated to theSphI/SalI-cut vector pUC18. The resulting plasmid was called pKB1321(FIG. 6).

pKB1321 was incubated with the restriction endonuclease HindII (suppliedby Boehringer Mannheim GmbH) under conditions which permit only partialcutting (0.05 U of enzymes per 1 μg of DNA for 10 min, other reactionconditions in accordance with manufacture's data). This produces, interalia, a fraction of fragments which is cut by HindII at positions 1793of the SerA gene. A DNA linker with an XbaI cleavage site was insertedat this point by ligation. The sequence of the DNA linker was as follow:

SEQ ID NO: 12

Insertion results in a PGD which carries 4 additional amino acids atthis point. Its sequence is depicted in Table 1. The plasmid with theinsertion was called pKB 1508 (FIG. 7). It was transformed into the serAmutant PC1523.

The serA allele serA11 was prepared by cutting the plasmid pGH5 withSalI and KpnI (supplied by Boehringer Mannheim GmbH) in accordance withthe manufacturer's data and purifying the fragment mixture after gelelectrophoresis of low-melting agarose. This fragment contains thevector portion from pUC18 and the C-terminal region from serA5. Theplasmid pKB 1508 was likewise cut with SalI/KpnI. The DNA fragment whichis 2.0 kb in size was eluted from a low-melting agarose gel. Thisfragment contains the serA allele serA1508 with the insertion mutation,but the 8 C-terminal codons are absent. The two fragments are ligatedtogether and used to transform the serA mutant PC1523. The resultingrecombinant plasmid was called pGH11 (FIG. 8). The encodedphosphoglycerate dehydrogenase combines the insertion mutation of serA1508 with the deletion mutation of serA5. The region with the mutationsin the encoded amino-acid sequence is shown in Table 1.

For expression of the mutated serA alleles in producer strains, theywere cloned into the vector pACYC 184 (ATCC37033), a vector with anintermediate copy number. To do this, the plasmid pGH5 and the plasmidpGH11 were cut with SalI/HindIII, and the DNA fragments which are eachabout 2 kb in size and which contain the serA alleles serA5 and serA11were isolated from low-melting agarose gels. The fragments were treatedin separate mixtures with the Klenow fragment of DNA polymerase I fromE. coli (supplied by Boehringer Mannheim GmbH) in accordance with themanufacturer's instructions in order to convert the 5′ protruding cutends of the restriction enzymes into blunt ends. To do this, 1 μg ofeach of the fragments was mixed in a 20 μl reaction mixture with 5 U ofKlenow enzyme, 0.5 mM dATP, dGTP, dTTP and dCTP and with the bufferrecommended by the manufacturer, and incubated at 30° C. for 15 min.

The blunt-ended DNA fragments are [sic] each ligated to a pACYC 184vector cut with PvuII. The ligation mixtures were used to transform theserA mutant PC1523. Complementing plasmids were called pGH5/II andpGH11/II respectively (FIG. 9, FIG. 10). The plasmid pKB1508 was cutwith SalI/SphI. The 3.0 kb fragment which contains the serA alleleserA1508 was purified by gel electrophoresis. The fragment was madeblunt-ended as described above and was ligated to PvuII-cut pACYC184,and the ligation mixture was transformed into E. coli PC1523.Complementing plasmids were called pKB1508/II (FIG. 11).

The plasmids pGH5/II (serA5), pGH11/II (serA11) and pKB1508/II were usedto transform the strains PD103 (trpE0), KB862 (trpE5), SV164 (trpE8) andSV163 (trpE6)

EXAMPLE 3 Construction of a Chromosomally Encoded, Feedback-ResistantserA5 Allele Using a Recombinant λ Prophage

For integration into the chromosomal lambda attachment site (att λ), theserA5 allele was cloned into the plasmid pRS551 (Simons et al., 1987,Gene 53: 85-96). To do this, the serA5-harbouring HindIII/SalI fragmentwhich is about 2 kb in size was isolated from the plasmid pGH5. The 5′protruding ends were filled in using the Klenow fragment of DNApolymerase I (supplied by Boehringer Mannheim GmbH) in accordance withthe manufacturer's data and, after attachment of EcoRI linkers (Maniatiset al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.: 396-397), the 2 kb fragment wasligated into the EcoRI-cleaved vector pRS551. A recombinant plasmid wasselected and called pRS5.

By preparing a plate lysate on a pRS5-harbouring recA⁺ strain (forexample YMC9 ATCC33927) with the λRS45 phage, a heterogeneous λ lysatewhich, besides λRS45 phages, also contained recombinant serA5-harbouringλRS45 derivatives was generated in vivo by homologous recombination(Simons et al., 1987, Gene 53: 85-96).

The serA strain PC1523 (CGSC#:5421) was used to select for recombinantλRS45 derivatives. To do this, PC1523 was infected with theheterogeneous λ lysate (see above) and subsequently plated out onkanamycin-containing. (25 mg/l) LB plates. The resulting lysogenickanamycin-resistant clones were then tested for their ability to grow onminimal medium plates without serine. A serine-prototrophic clone wasselected and used to prepare a homogeneous serA5 λ lysate (by UVinduction Simons et al., 1987, Gene 53: 85-96).

This homogeneous serA5 λ lysate was used to infect the tryptophanproducer strain SV164. The resulting strain SV164 attλ::serA5 wasfermented as described in Example 4. The particular media contained inplace of tetracycline as selecting agent in each case 25 mg/l kanamycin.

The tryptophan yields were around 12.5 g/l, compared with 3.5 g/l usingthe same strain without serA5.

EXAMPLE 4 Tryptophan Production Using Corynebacteria

The plasmid pGH5 is cut with the restriction enzymes SalI and HindIII(supplied by Boehringer Mannheim GmbH), and the DNA fragment which is 2kb in size and harbours the serA5 gene is isolated from a low-meltingagarose gel. The DNA fragment is made blunt-ended by the action of theKlenow fragment of DNA polymerase I from E. coli (supplied by BoehringerMannheim GmbH) as described in Example 2. The vector pWST1 is cut withthe restriction enzyme SmaI (supplied by Boehringer) and ligated to theblunt-ended DNA fragment. The vector pWST1 is an E. coli/corynebacteriashuttle vector and can replicate both in E. coli and in corynebacteria.The corynebacterial replicon of this vector is derived from the strainCorynebacterium glutamicum ATCC 19223. The preparation of the vectorpWST1 is described in U.S. Pat. No. 4,965,197. The ligation mixture isused to transform the E. coli strain PC1523. Complementing plasmids arecalled pGH5/III (FIG. 12).

The plasmid pGH5/III is used to transform the tryptophan-producingCorynebacterium glutamicum ATCC21851. The transformation is carried outby electrophoration [sic] by the technique described in detail by WolfH. et al., 1989, Appl. Microbiol. Biotechnol. 30: 283-289. Clones whichharbour the recombinant plasmid pGH5/III are selected via theplasmid-encoded kanamycin resistance on agar plates containing 25 mg/lkanamycin.

The plasmid pGC3 is cut with the restriction enzymes SphI and SalI. The3 kb DNA fragment which harbours the serA wild-type allele is purifiedand ligated into the vector pWST1 in the manner described above. Theresulting vector pGC3/I (FIG. 13) is used to transform Corynebacteriumglutamicum ATCC21851.

A Corynebacterium glutamicum ATCC21581 strain which harbours the serAallele 1455 on a plasmid is prepared analogously.

Fermentation reveals that the strain which harbours the serA5 allele ona plasmid achieves the highest tryptophan yields.

EXAMPLE 5 Effect of Various Plasmid-Encoded serA Alleles on theTryptophan Production of Various trpE Strains

10 ml of LB medium (1% Tryptone, 0.5% yeast extract, 0.5% NaCl), towhich 15 mg/l tetracycline were added, in a 100 ml conical flask wereinoculated with the various tryptophan producer strains summarised inTable 3. After incubation at 30° C., shaking at 150 rpm, for 8-9 hours,the particular precultures were transferred into 100 ml of SM1 medium.The SM1 medium contained 5 g/l glucose, 3 g/l KH₂PO₄, 12 g/l K₂HPO₄, 0.1g/l (NH₄)₂SO₄, 0.3 g/l MgSO₄×7H₂O, 15 mg/l CaCl₂×2 H₂O, 2 mg/l FeSO₄×7H₂O, 1 g/l Na₃ citrate×2H₂O, 1 ml/l trace element solution (see below),40 mg/l L-phenylalanine, 40 mg/l L-tyrosine, 5 mg/l vitamin B1 and 15mg/l tetracycline. The trace element solution was composed of 0.15 g/lNa₂MoO₄×2H₂O, 2.5 g/l H₃BO₃, 0.7 g/l CoCl₂×6H₂O, , 0.25 g/l CuSO₄×5H₂O,1.6 g/l MnCl₂×4H₂O and 0.3 g/l ZnSO₄×7H₂O. The cultures were shaken at150 rpm in 1 l conical flasks at 30° C. for 12-16 h. The OD₆₀₀ afterthis incubation was between 2 and 4. Further fermentation is [sic]carried out in BIOSTAT®M research fermenters supplied byBraun-Melsungen. A culture vessel with a total volume of 2 liters wasused.

The medium contained 17.5 g/1 glucose, 5 g/l (NH₄)₂SO₄, 0.5 g/l NaCl,0.3 g/l MgSO₄×7H₂O, 15 mg/l CaCl₂×2H₂O, 75 mg/l FeSO₄×7H₂O, 1 g/l Na₃citrate×2 H₂O, 1.5 g/l KH₂PO₄, 1 ml trace element solution (see above),5 mg/l vitamin B1 (thiamine), 0.75 g/l L-phenylalanine, 0.75 g/lL-tyrosine, 2.5 g/l yeast extract (Difco), 2.5 g/l Tryptone (Difco) and20 mg/l tetracycline.

The glucose concentration in the fermenter was adjusted to 17.5 g/l bypumping in a 700 g/l (w/v) glucose solution (autoclaved). Beforeinoculation, tetracycline was added to a final concentration of 20 mg/lin the fermentation medium. In addition, the pH was adjusted to 6.7 bypumping in 25% NH₄OH solution.

100 ml of preculture were pumped into the fermentation vessel forinoculation. The initial volume was about 1 l. The cultures wereinitially stirred at 400 rpm, and compressed air sterilised with asterilising filter was passed in at 1.5 vvm. The fermentation wascarried out at a temperature of 30° C.

The pH was kept at a value of 6.7 by automatic correction with 25%NH₄OH. The oxygen saturation in the fermentation broth ought not to fallbelow 20% at any time during the fermentation. The oxygen saturation wascontrolled via the stirring speed during the fermentation.

At intervals of two to three hours, the glucose content of the nutrientsolution, the optical density and the tryptophan yield were measured.The glucose content was determined enzymatically using a glucoseanalyser supplied by YSI. The glucose concentration was adjusted tobetween 5 and 20 g/l by continuous feeding in.

The tryptophan content of the medium after the fermentation wasdetermined by HPLC. The medium was fractionated on a Nucleosil 100-7/C8[lacuna] (250/4 mm; Macherey-Nagel). The column was operatedisocractically at a flow rate of 2 ml/min. The mobile phase used waswater/acetonitrile (83/17) to which 0.1 ml of H₃PO₄ (85%) was added perliter. Detection was carried out either with a diode array detector orat a fixed wavelength of 215 or 275 nm. The fermentation was stoppedafter 44-50 h. The amounts of tryptophan produced in this fermentationin g/l after 48 h are summarised in Table 3.

TABLE 3 Tryptophan yields with various serA/trpE combinations serAWTserA5 serA1508 serA11 serA1455 trpE0 15.7 20.2 n.d. n.d. 6.7 trpE5 12.518.9 15.0 20.0 7.5 trpE6 11.6 24.1 13.8 24.0 4.0 trpE8  7.5 18.0 n.d.11.5 3.9 n.d. not determined

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES:  14(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  52 amin #o acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein   (iii) HYPOTHETICAL:  YES      (v) FRAGMENT TYPE: terminal fragment    (vi) ORIGINAL SOURCE:           (A) ORGANISM:  Escheric #hia coli          (B) STRAIN:  B    (vii) IMMEDIATE SOURCE:          (B) CLONE:  pGC3     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO:#1: Ala Glu Gln Gly Val Asn Ile Ala Ala Gln Ty #r Leu Gln Thr Ser Ala1               5    #                10   #                15Gln Met Gly Tyr Val Val Ile Asp Ile Glu Al #a Asp Glu Asp Val Ala            20       #            25       #            30Glu Lys Ala Leu Gln Ala Met Lys Ala Ile Pr #o Gly Thr Ile Arg Ala        35           #        40           #        45 Arg Leu Leu Tyr    50 (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  51 amin#o acids           (B) TYPE:    Amin #o acid          (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY:  linear    (ii) MOLECULE TYPE:  protein    (iii) HYPOTHETICAL:  YES     (v) FRAGMENT TYPE: C terminal fragmen #t     (vi) ORIGINAL SOURCE:          (A) ORGANISM:  Escheric #hia coli           (B) STRAIN:  B   (vii) IMMEDIATE SOURCE:           (B) CLONE:  pGH5    (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #2:Ala Glu Gln Gly Val Asn Ile Ala Ala Gln Ty #r Leu Gln Thr Ser Ala1               5    #                10   #                15Gln Met Gly Tyr Val Val Ile Asp Ile Glu Al #a Asp Glu Asp Val Ala            20       #            25       #            30Glu Lys Ala Leu Gln Ala Met Lys Ala Ile Pr #o Gly Thr Ile Arg Ala        35           #        40           #        45 Arg Leu Leu    50 (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  56 amin#o acids           (B) TYPE:    Amin #o acid          (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY:  linear    (ii) MOLECULE TYPE:  protein    (iii) HYPOTHETICAL:  YES     (v) FRAGMENT TYPE: C terminal fragmen #t     (vi) ORIGINAL SOURCE:          (A) ORGANISM:  Escheric #hia coli           (B) STRAIN:  B   (vii) IMMEDIATE SOURCE:           (B) CLONE:  pKB1508    (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #3:Ala Glu Gln Gly Val Cys Ser Arg Ala Asn Il #e Ala Ala Gln Tyr Leu1               5    #                10   #                15Gln Thr Ser Ala Gln Met Gly Tyr Val Val Il #e Asp Ile Glu Ala Asp            20       #            25       #            30Glu Asp Val Ala Glu Lys Ala Leu Gln Ala Me #t Lys Ala Ile Pro Gly        35           #        40           #        45Thr Ile Arg Ala Arg Leu Leu Tyr     50               #    55(2) INFORMATION FOR SEQ ID NO:4:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  55 amin #o acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein   (iii) HYPOTHETICAL:  YES      (v) FRAGMENT TYPE: C terminal fragmen#t     (vi) ORIGINAL SOURCE:           (A) ORGANISM:  Escheric #hia coli          (B) STRAIN:  B    (vii) IMMEDIATE SOURCE:          (B) CLONE:  pGH11     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO:#4: Ala Glu Gln Gly Val Cys Ser Arg Ala Asn Il #e Ala Ala Gln Tyr Leu1               5    #                10   #                15Gln Thr Ser Ala Gln Met Gly Tyr Val Val Il #e Asp Ile Glu Ala Asp            20       #                25   #            30Glu Asp Val Ala Glu Lys Ala Leu Gln Ala Me #t Lys Ala Ile Pro Gly        35           #        40           #        45Thr Ile Arg Ala Arg Leu Leu     50               #    55(2) INFORMATION FOR SEQ ID NO:5:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  47 amin #o acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein   (iii) HYPOTHETICAL:  YES      (v) FRAGMENT TYPE: C terminal fragmen#t     (vi) ORIGINAL SOURCE:           (A) ORGANISM:  Escheric #hia coli          (B) STRAIN:  B    (vii) IMMEDIATE SOURCE:          (B) CLONE:  pKB1455     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO:#5: Ala Glu Gln Gly Val Asn Ile Ala Ala Gln Ty #r Leu Gln Thr Ser Ala1               5    #                10   #                15Gln Met Gly Tyr Val Val Ile Asp Ile Glu Al #a Asp Glu Asp Val Ala            20       #            25       #            30Glu Lys Ala Leu Gln Ala Met Lys Ala Ile Pr #o Gly Thr Ile Arg        35           #        40           #        45(2) INFORMATION FOR SEQ ID NO:6:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  32 amin #o acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein   (iii) HYPOTHETICAL:  YES      (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:           (A) ORGANISM:  Escheric #hia coli          (B) STRAIN: YMC9     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO:#6: Asn Pro Thr Ala Leu Phe His Gln Leu Cys Gl #y Asp Arg Pro Ala Thr1               5    #                10   #                15Leu Leu Leu Glu Ser Ala Asp Ile Asp Ser Ly #s Asp Asp Leu Lys Ser            20       #            25       #            30(2) INFORMATION FOR SEQ ID NO:7:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  32 amin #o acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein   (iii) HYPOTHETICAL:  YES    (iii) ANTI-SENSE:    NO     (v) FRAGMENT TYPE: internal fragment     (vi) ORIGINAL SOURCE:          (A) ORGANISM:  Escheric #hia coli           (B) STRAIN:  PD103    (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #7:Asn Pro Thr Ala Leu Phe His Gln Leu Cys Gl #y Asp Arg Pro Ala Thr1               5    #                10   #                15Leu Leu Leu Glu Ser Ala Asp Ile Asp Ser Ly #s Asp Asp Leu Glu Ser            20       #            25       #            30(2) INFORMATION FOR SEQ ID NO:8:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  32 amin #o acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein   (iii) HYPOTHETICAL:  YES      (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:           (A) ORGANISM:  Escheric #hia coli          (B) STRAIN:  KB862     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO:#8: Asn Ser Thr Ala Leu Phe His Gln Leu Cys Gl #y Asp Arg Pro Ala Thr1               5    #                10   #                15Leu Leu Leu Glu Ser Ala Asp Ile Asp Ser Ly #s Asp Asp Leu Lys Ser            20       #            25       #            30(2) INFORMATION FOR SEQ ID NO:9:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  32 amin #o acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein   (iii) HYPOTHETICAL:  YES      (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:           (A) ORGANISM:  Escheric #hia coli          (B) STRAIN:  SV163     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO:#9: Asn Pro Thr Ala Leu Phe His Gln Leu Cys Gl #y Asp Arg Pro Ala Thr1               5    #                10   #                15Leu Leu Leu Glu Phe Ala Asp Ile Asp Ser Ly #s Asp Asp Leu Glu Ser            20       #            25       #            30(2) INFORMATION FOR SEQ ID NO:10:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  32 amin #o acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein   (iii) HYPOTHETICAL:  YES      (v) FRAGMENT TYPE: internal fragment    (vi) ORIGINAL SOURCE:           (A) ORGANISM:  Escheric #hia coli          (B) STRAIN:  SV164     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO:#10: Asn Ser Thr Ala Leu Phe His Gln Leu Cys Gl #y Asp Arg Pro Ala Thr1               5    #                10   #                15Leu Leu Leu Glu Ser Ala Asp Ile Asp Ser Ly #s Asp Asp Leu Glu Ser            20       #            25       #            30(2) INFORMATION FOR SEQ ID NO:11:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  32 base # pairs           (B) TYPE:    Nucl#eic acid           (C) STRANDEDNESS:  both          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  DNA   (iii) HYPOTHETICAL:  NO    (iii) ANTI-SENSE:  NO    (vi) ORIGINAL SOURCE:           (A) ORGANISM:  DNA f#ragment synthesized in vitro     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO:#11: AGCTTATTAC AGCAGACGGG CGCGAATGGA TC        #                  #          32 (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  12 base# pairs           (B) TYPE:    Nucl #eic acid          (C) STRANDEDNESS:  doub #le           (D) TOPOLOGY:  linear    (ii) MOLECULE TYPE:  DNA    (iii) HYPOTHETICAL:  NO   (iii) ANTI-SENSE:   NO     (vi) ORIGINAL SOURCE:          (A) ORGANISM:  DNA f #ragment synthesized in vitro    (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #12:TGCTCTAGAG CA               #                   #                  #       12 (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  1232 ba#se pairs           (B) TYPE:    Nucl #eic acid          (C) STRANDEDNESS:  doub #le           (D) TOPOLOGY:  linear    (ii) MOLECULE TYPE:  DNA (genomic)    (iii) HYPOTHETICAL:  NO   (iii) ANTI-SENSE:  NO     (vi) ORIGINAL SOURCE:          (A) ORGANISM:  Escheric #hia coli           (B) STRAIN:  B    (ix) FEATURE:           (A) NAME/KEY:  CDS          (B) LOCATION:  1..1233           (C) IDENTIFICATION METHOD:#  experimental           (D) OTHER INFORMATION:  # /coden_start=1               /EC_ #numer=1.1.1.95                /product=# “D-3-Phosphoglycerate-                dehydrogenas #e”               /evidence:  #EXPERIMENTAL                /gen=  #“serA”               /standard_ #name= “serA”                /citation=# ([1])      (x) PUBLICATION INFORMATION:           (A) AUTHORS:     #  Tobey, K.L.                    #           Grant, G.A.          (B) TITLE: The nucleoti #de sequence of the               serA gene # of Escherichia coli and               the amino # acid sequence of the                encoded p#rotein, d-3-phospho-                glycerate  #dehydrogenase          (C) JOURNAL:  J. Bio #l. Chem.           (D) VOLUME:   261          (F) PAGES:    121 #79-12183           (G) DATE:      #1986    (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #13:ATG GCA AAG GTA TCG CTG GAG AAA GAC AAG AT#T AAG TTT CTG CTG GTA       48Met Ala Lys Val Ser Leu Glu Lys Asp Lys Il #e Lys Phe Leu Leu Val1               5    #                10   #                15GAA GGC GTG CAC CAA AAG GCG CTG GAA AGC CT#T CGT GCA GCT GGT TAC       96Glu Gly Val His Gln Lys Ala Leu Glu Ser Le #u Arg Ala Ala Gly Tyr            20       #            25       #            30ACC AAC ATC GAA TTT CAC AAA GGC GCG CTG GA#T GAT GAA CAA TTA AAA      144Thr Asn Ile Glu Phe His Lys Gly Ala Leu As #p Asp Glu Gln Leu Lys        35           #        40           #        45GAA TCC ATC CGC GAT GCC CAC TTC ATC GGC CT#G CGA TCC CGT ACC CAT      192Glu Ser Ile Arg Asp Ala His Phe Ile Gly Le #u Arg Ser Arg Thr His    50               #    55               #    60CTG ACT GAA GAC GTG ATC AAC GCC GCA GAA AA#A CTG GTC GCT ATT GGC      240Leu Thr Glu Asp Val Ile Asn Ala Ala Glu Ly #s Leu Val Ala Ile Gly65                   #70                   #75                   #80TGT TTC TGT ATC GGA ACA AAC CAG GTT GAT CT#G GAT GCG GCG GCA AAG      288Cys Phe Cys Ile Gly Thr Asn Gln Val Asp Le #u Asp Ala Ala Ala Lys                85   #                90   #                95CGC GGG ATC CCG GTA TTT AAC GCA CCG TTC TC#A AAT ACG CGC TCT GTT      336Arg Gly Ile Pro Val Phe Asn Ala Pro Phe Se #r Asn Thr Arg Ser Val            100       #           105       #           110GCG GAG CTG GTG ATT GGC GAA CTG CTG CTG CT#A TTG CGC GGC GTG CCG      384Ala Glu Leu Val Ile Gly Glu Leu Leu Leu Le #u Leu Arg Gly Val Pro        115           #       120           #       125GAA GCC AAT GCT AAA GCG CAC CGT GGC GTG TG#G AAC AAA CTG GCG GCG      432Glu Ala Asn Ala Lys Ala His Arg Gly Val Tr #p Asn Lys Leu Ala Ala    130               #   135               #   140GGT TCT TTT GAA GCG CGC GGC AAA AAG CTG GG#T ATC ATC GGC TAC GGT      480Gly Ser Phe Glu Ala Arg Gly Lys Lys Leu Gl #y Ile Ile Gly Tyr Gly145                 1 #50                 1 #55                 1 #60CAT ATT GGT ACG CAA TTG GGC ATT CTG GCT GA#A TCG CTG GGA ATG TAT      528His Ile Gly Thr Gln Leu Gly Ile Leu Ala Gl #u Ser Leu Gly Met Tyr                165   #               170   #               175GTT TAC TTT TAT GAT ATT GAA AAT AAA CTG CC#G CTG GGC AAC GCC ACT      576Val Tyr Phe Tyr Asp Ile Glu Asn Lys Leu Pr #o Leu Gly Asn Ala Thr            180       #           185       #           190CAG GTA CAG CAT CTT TCT GAC CTG CTG AAT AT#G AGC GAT GTG GTG AGT      624Gln Val Gln His Leu Ser Asp Leu Leu Asn Me #t Ser Asp Val Val Ser        195           #       200           #       205CTG CAT GTA CCA GAG AAT CCG TCC ACC AAA AA#T ATG ATG GGC GCG AAA      672Leu His Val Pro Glu Asn Pro Ser Thr Lys As #n Met Met Gly Ala Lys    210               #   215               #   220GAA ATT TCA CTA ATG AAG CCC GGC TCG CTG CT#G ATT AAT GCT TCG CGC      720Glu Ile Ser Leu Met Lys Pro Gly Ser Leu Le #u Ile Asn Ala Ser Arg225                 2 #30                 2 #35                 2 #40GGT ACT GTG GTG GAT ATT CCG GCG CTG TGT GA#T GCG CTG GCG AGC AAA      768Gly Thr Val Val Asp Ile Pro Ala Leu Cys As #p Ala Leu Ala Ser Lys                245   #               250   #               255CAT CTG GCG GGG GCG GCA ATC GAC GTA TTC CC#G ACG GAA CCG GCG ACC      816His Leu Ala Gly Ala Ala Ile Asp Val Phe Pr #o Thr Glu Pro Ala Thr            260       #           265       #           270AAT AGC GAT CCA TTT ACC TCT CCG CTG TGT GA#A TTC GAC AAC GTC CTT      864Asn Ser Asp Pro Phe Thr Ser Pro Leu Cys Gl #u Phe Asp Asn Val Leu        275           #       280           #       285CTG ACG CCA CAC ATT GGC GGT TCG ACT CAG GA#A GCG CAG GAG AAT ATC      912Leu Thr Pro His Ile Gly Gly Ser Thr Gln Gl #u Ala Gln Glu Asn Ile    290               #   295               #   300GGC CTG GAA GTT GCG GGT AAA TTG ATC AAG TA#T TCT GAC AAT GGC TCA      960Gly Leu Glu Val Ala Gly Lys Leu Ile Lys Ty #r Ser Asp Asn Gly Ser305                 3 #10                 3 #15                 3 #20ACG CTC TCT GCG GTG AAC TTC CCG GAA GTC TC#G CTG CCA CTG CAC GGT     1008Thr Leu Ser Ala Val Asn Phe Pro Glu Val Se #r Leu Pro Leu His Gly                325   #               330   #               335GGG CGT CGT CTG ATG CAC ATC CAC GAA AAC CG#T CCG GGC GTG CTA ACT     1056Gly Arg Arg Leu Met His Ile His Glu Asn Ar #g Pro Gly Val Leu Thr            340       #           345       #           350GCG CTG AAC AAA ATC TTC GCC GAG CAG GGC GT#C AAC ATC GCC GCG CAA     1104Ala Leu Asn Lys Ile Phe Ala Glu Gln Gly Va #l Asn Ile Ala Ala Gln        355           #       360           #       365TAT CTG CAA ACT TCC GCC CAG ATG GGT TAT GT#G GTT ATT GAT ATT GAA     1152Tyr Leu Gln Thr Ser Ala Gln Met Gly Tyr Va #l Val Ile Asp Ile Glu    370               #   375               #   380GCC GAC GAA GAC GTT GCC GAA AAA GCG CTG CA#G GCA ATG AAA GCT ATT     1200Ala Asp Glu Asp Val Ala Glu Lys Ala Leu Gl #n Ala Met Lys Ala Ile385                 3 #90                 3 #95                 4 #00CCG GGT ACC ATT CGC GCC CGT CTG CTG TAC TA #                  #        1232 Pro Gly Thr Ile Arg Ala Arg Leu Leu Tyr                405   #               410(2) INFORMATION FOR SEQ ID NO:14:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH:  410 ami #no acids           (B) TYPE:    Amin#o acid           (C) STRANDEDNESS:  sing #le          (D) TOPOLOGY:  linear     (ii) MOLECULE TYPE:  protein    (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #14:Met Ala Lys Val Ser Leu Glu Lys Asp Lys Il #e Lys Phe Leu Leu Val1               5    #                10   #                15Glu Gly Val His Gln Lys Ala Leu Glu Ser Le #u Arg Ala Ala Gly Tyr            20       #            25       #            30Thr Asn Ile Glu Phe His Lys Gly Ala Leu As #p Asp Glu Gln Leu Lys        35           #        40           #        45Glu Ser Ile Arg Asp Ala His Phe Ile Gly Le #u Arg Ser Arg Thr His    50               #    55               #    60Leu Thr Glu Asp Val Ile Asn Ala Ala Glu Ly #s Leu Val Ala Ile Gly65                   #70                   #75                   #80Cys Phe Cys Ile Gly Thr Asn Gln Val Asp Le #u Asp Ala Ala Ala Lys                85   #                90   #                95Arg Gly Ile Pro Val Phe Asn Ala Pro Phe Se #r Asn Thr Arg Ser Val            100       #           105       #           110Ala Glu Leu Val Ile Gly Glu Leu Leu Leu Le #u Leu Arg Gly Val Pro        115           #       120           #       125Glu Ala Asn Ala Lys Ala His Arg Gly Val Tr #p Asn Lys Leu Ala Ala    130               #   135               #   140Gly Ser Phe Glu Ala Arg Gly Lys Lys Leu Gl #y Ile Ile Gly Tyr Gly145                 1 #50                 1 #55                 1 #60His Ile Gly Thr Gln Leu Gly Ile Leu Ala Gl #u Ser Leu Gly Met Tyr                165   #               170   #               175Val Tyr Phe Tyr Asp Ile Glu Asn Lys Leu Pr #o Leu Gly Asn Ala Thr            180       #           185       #           190Gln Val Gln His Leu Ser Asp Leu Leu Asn Me #t Ser Asp Val Val Ser        195           #       200           #       205Leu His Val Pro Glu Asn Pro Ser Thr Lys As #n Met Met Gly Ala Lys    210               #   215               #   220Glu Ile Ser Leu Met Lys Pro Gly Ser Leu Le #u Ile Asn Ala Ser Arg225                 2 #30                 2 #35                 2 #40Gly Thr Val Val Asp Ile Pro Ala Leu Cys As #p Ala Leu Ala Ser Lys                245   #               250   #               255His Leu Ala Gly Ala Ala Ile Asp Val Phe Pr #o Thr Glu Pro Ala Thr            260       #           265       #           270Asn Ser Asp Pro Phe Thr Ser Pro Leu Cys Gl #u Phe Asp Asn Val Leu        275           #       280           #       285Leu Thr Pro His Ile Gly Gly Ser Thr Gln Gl #u Ala Gln Glu Asn Ile    290               #   295               #   300Gly Leu Glu Val Ala Gly Lys Leu Ile Lys Ty #r Ser Asp Asn Gly Ser305                 3 #10                 3 #15                 3 #20Thr Leu Ser Ala Val Asn Phe Pro Glu Val Se #r Leu Pro Leu His Gly                325   #               330   #               335Gly Arg Arg Leu Met His Ile His Glu Asn Ar #g Pro Gly Val Leu Thr            340       #           345       #           350Ala Leu Asn Lys Ile Phe Ala Glu Gln Gly Va #l Asn Ile Ala Ala Gln        355           #       360           #       365Tyr Leu Gln Thr Ser Ala Gln Met Gly Tyr Va #l Val Ile Asp Ile Glu    370               #   375               #   380Ala Asp Glu Asp Val Ala Glu Lys Ala Leu Gl #n Ala Met Lys Ala Ile385                 3 #90                 3 #95                 4 #00Pro Gly Thr Ile Arg Ala Arg Leu Leu Tyr                 405  #               410

What is claimed is:
 1. A tryptophan producing strain of microorganism,said tryptophan producing strain of microorganism being selected fromthe group consisting of E. coli and Corynebacteria and is tryptophanfeedback resistant and serine feedback resistant and wherein said serinefeedback resistance is by a mutation in a serA allele, where the mutatedserA allele codes for a protein which has a K_(i) value for serinebetween 0.1 mM and 50 mM; and wherein said tryptophan feedbackresistance is by a trpE allele which codes for a protein which has aK_(i) value for tryptophan between 0.1 mM and 20 mM.
 2. The strainaccording to claim 1, wherein the serA allele is integrated into thechromosome.
 3. The strain according to claim 1, wherein the bacteriabelong to the species E. coli.
 4. The strain according to claim 1,wherein the bacteria belong to the species Corynebacteria.
 5. The strainaccording to claim 1, wherein the serA allele mutation has a C-terminalamino acid sequence selected from the group consisting of: (SEQ IDNO:2); (SEQ ID NO:3); (SEQ ID NO:4); and (SEQ ID NO:5).
 6. The strainaccording to claim 1, wherein said trpE allele has an amino acidsequence selected from the group consisting of (SEQ ID NO:7); (SEQ IDNO:8); (SEQ ID NO:9); and (SEQ ID NO:10).
 7. The strain according toclaim 1, wherein the serA allele mutation has a C-terminal amino acidsequence selected from the group consisting of: (SEQ ID NO:2); (SEQ IDNO:3); (SEQ ID NO:4); (SEQ ID NO:5); and wherein said trpE allele has anamino acid sequence selected from the group consisting of (SEQ ID NO:7);(SEQ ID NO:8); (SEQ ID NO:9); and (SEQ ID NO:10).
 8. Process for thepreparation of a tryptophan producing strain of microorganism comprisingproviding a tryptophan producing strain of microorganism selected fromthe group consisting of E. coli and Corynebacteria with a tryptophanfeedback resistance; introducing a serine feedback-resistance serAallele into said microorganism strain with said tryptophan feedbackresistance; and wherein said serine feedback-resistance is by a mutationin a serA allele, where the mutated serA allele codes for a proteinwhich has a K_(i) value for serine between 0.1 mM and 50 mM; and whereinsaid tryptophan feedback resistance is by a trpE allele which codes fora protein which has a K_(i) value for tryptophan between 0.1 mM and 20mM.
 9. Process according to claim 8, comprising introducing the serAallele into the chromosome of the strain of microorganism with saidtryptophan feedback resistance.
 10. In a method for producing tryptophancomprising culturing a tryptophan producing strain of microorganism in aculture medium; and recovering the produced tryptophan from the culturemedium; the improvement which comprises utilizing a tryptophan producingstrain of microorganism selected from the group consisting of E. coliand Corynebacteria which is tryptophan feedback resistant and serinefeedback resistant and wherein said serine feedback resistance is by amutation in a serA allele, where the mutated serA allele codes for aprotein which has a K_(i) value for serine between 0.1 mM and 50 mM toproduce said tryptophan; and wherein said tryptophan feedback resistanceis by a trpE allele which codes for a protein which has a K_(i) valuefor tryptophan between 0.1 mM and 20 mM.